Transcriptional regulation of mitochondrial glycerophosphate acyltransferase is mediated by distal promoter via ChREBP and SREBP-1

https://doi.org/10.1016/j.abb.2009.07.027Get rights and content

Abstract

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.

Introduction

Glycerol-3-phosphate acyltransferase (GPAT)4 converts sn-glycerol-3-phosphate to 1-acyl-sn-glycerol-3-phosphate, the first step in the biosynthetic pathway of all glycerolipids. Mitochondrial GPAT (mtGPAT) is an enzyme that can switch the fate of fatty acids from β-oxidation to glycerolipid synthesis [1], [2]. The mtGPAT isoform is known to control the asymmetric distribution of fatty acids in cellular glycerolipids. The asymmetric distribution of fatty acids plays an important role in maintaining structure and function of phospholipids present in cellular membranes [3]. The mitochondrial acyltransferase is known to comprise ∼10% of total GPAT activity in most tissues except in the liver where it comprises ∼50% of the total GPAT activity [4]. Hepatic mtGPAT mRNA level is up-regulated by a high carbohydrate, fat-free diet and by insulin administration to streptozotocin-diabetic mice. Also, mtGPAT activity is higher in the livers from both diet-induced obese mice and leptin-deficient ob/ob mice when compared with their lean controls [5].

It has been shown that elevated hepatic mtGPAT is associated with obesity since the enzyme can divert activated fatty acids from oxidation to glycerolipid synthesis. Thus, the increase in mtGPAT activity could be at least partly responsible for obesity and lipid disorders associated with obesity [2]. Therefore, understanding the molecular mechanism of mtGPAT transcriptional regulation is important.

We have previously shown the presence of two promoters for rat mtGPAT: a distal promoter which is ∼30 kb upstream of the first translational codon and a proximal promoter which is 63 bp upstream of the second translational codon. It is known that lipogenic enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), like mtGPAT, are hormonally and nutritionally regulated – properties consistent with their role in triacylglycerol synthesis [6]. Studies show that insulin and EGF stimulate lipogenesis and ACC activity in isolated adipocytes [7]. On the other hand, leptin and glucagon are known to inhibit lipogenic enzyme synthesis by acting through decrease of the amounts of the lipogenic transcription factor, SREBP-1c and intracellular mediator cAMP, respectively [2], [6]. In an effort to determine whether the distal or the proximal promoter is responsible for the transcriptional regulation of rat mtGPAT, two stimulating agents (insulin and EGF) and two inhibiting agents (glucagon and leptin) for lipogenesis were used in this investigation.

The transcriptional factors such as ChREBP (100 kDa) and SREBP-1c (68 kDa) are known to be major regulators of lipogenic enzymes [8], [9]. ChREBP is predominantly expressed in liver, kidney, white and brown adipose tissues and is known to recognize E box sequences in the promoters of target genes and is predominantly present in inactive phosphorylated form in the cytoplasm [10]. The regulation of ChREBP is relatively simple and efficient, and involves phosphorylation-dependent mechanisms responsive to feeding (glucose and fatty acids) and fasting (glucagon) [8]. The extent of phosphorylation of ChREBP determines its nuclear or cytoplasmic location. For example, when overnight-fasted mice were refed a high-carbohydrate diet for 18 h, there was low ChREBP phosphorylation on Ser196 causing it to be predominantly located in the nucleus. When glucagon was injected (0.5 U/kg) into the portal vein of refed mice, ChREBP phosphorylation on Ser196 was increased and ChREBP protein was exported from the nucleus [11]. Three SREBPs have been identified so far, SREBP-1a and SREBP-1c are encoded from the same gene while SREBP-2 is encoded from a separate gene. The predominant SREBP-1 isoform in liver and adipose tissue is 1c rather than 1a [12]. Hence in all likelihood, SREBP-1c actually binds to the distal promoter due to its abundance in liver cells. Promoter inspector analysis showed the presence of the binding sites for SREBP-1 and ChREBP only in the distal promoter of rat mtGPAT gene. There is no such presence of lipogenic transcription factor binding sites in the proximal promoter region [13]. Leptin is known to decrease expression of lipogenic enzyme gene through modification in the SREBP-1c gene expression [14]. EGF stimulates SREBP-1c mRNA levels in prostate cancer cells and causes an increase in mRNA levels of the lipogenic enzyme FAS [15]. Studies have shown that the negative target for leptin is SREBP-1, suggesting that this transcription factor may be involved in mediating the inhibitory effect of leptin on lipogenic gene expression [16], [17]. Insulin was found to increase the levels of ChREBP mRNA in 3T3 L1 adipocytes and to promote lipogenesis in newly formed adipocytes [18].

In this study, we have investigated the role of the two promoters in transcriptional regulation of rat mtGPAT gene expression. We present evidence that the distal promoter is the regulatory promoter under the influence of hormones and growth factors while the proximal promoter acts like a constitutive promoter. To gain further insight into the molecular mechanism of how the distal promoter regulates the mtGPAT gene transcription, we have assessed the role of lipogenic transcription factors, ChREBP and SREBP-1c by means of electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP).

Section snippets

Cell culture

Buffalo rat liver cells (BRL) which are hepatocytes were bought from ATCC (CRL-1442) and were cultured in Eagle’s minimum essential medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 50 U/ml of penicillin–streptomycin at 37 °C in a humidified atmosphere of 5% CO2.

Chemicals

Insulin, EGF, glucagon, leptin, formaldehyde and protease inhibitors were purchased from Sigma–Aldrich; penicillin–streptomycin was from Gibco; wortmannin and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) were from

The distal 520 bp mtGPAT promoter is essential for its regulatory effect

A schematic diagram of the distal and the proximal promoters with putative transcription factor binding sites, TATA box and inverted CAAT box is presented in Fig. 1A. We have previously shown that 500 bp of the proximal promoter region has about 2-fold luciferase activity over the full-length 1050 bp region [13]. Similarly, transfection experiments were performed to define the region of the distal promoter that is required for its maximum promoter activity. A series of 5′-nested serial deletion

Discussion

The present study provides insight into the mechanism of the transcriptional regulation of rat mtGPAT gene. We have demonstrated here that the distal promoter is the regulatory promoter under the influence of hormones and growth factor while the proximal promoter acts constitutively. We have further investigated the mechanism of the transcriptional regulation by the distal promoter and have determined that the distal promoter controls the transcriptional regulation by lipogenic transcription

Acknowledgments

We would like to thank Dr. Ales Vancura for his help. This research was supported by the National Institutes of Health Grant GM-57643 (D.H.) and by Clare Boothe Luce fellowship (K.K.A.). This work forms a portion of a doctoral thesis (P.G.) submitted to the faculty of Biological Sciences, St. John’s University.

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